Batch and Flow-Injection Spectrophotometric Determination of Procaine HCl in Pharmaceutical Preparations Via Using Diazotization and Coupling Reaction

: Simple and sensitive batch and Flow-injection spectrophotometric methods for the determination of Procaine HCl in pure form and in injections were proposed. These methods were based on a diazotization reaction of procaine HCl with sodium nitrite and hydrochloric acid to form diazonium salt, which is coupled with chromatropic acid in alkaline medium to form an intense pink water-soluble dye that is stable and has a maximum absorption at 508 nm. A graphs of absorbance versus concentration show that Beer’s law is obeyed over the concentration range of 1-40 and 5-400 µg.ml - 1 of Procaine HCl, with detection limits of 0.874 and 3.75 µg.ml -1 of Procaine HCl for batch and FIA methods respectively. The FIA average sample throughput was 70 h -1 . All different chemical and physical experimental parameters that affecting on the development and stability of the colored product were carefully studied and the proposed methods were applied satisfactorily for the determination of Procaine HCl in an injections samples using the standard addition method.


Introduction:
Procaine is a local anesthetic can be used either alone or with penicillin as an antibacterial drug its chemical name is 2-diethyl aminoethyl-4aminobenzoate hydrochloride and its formula C 13 H 20 N 2 O 2 , with molecular weight of 272.8 the structure of procaine HCl is [1]: Procaine was first synthesized in 1905, it quickly replaced cocaine because it is easier to synthesize and sterilize, shorter duration of action, non addictive and it has four to six times less toxic than cocaine, procaine, like other local anesthetics such as tetracaine acts as a nerve block, halting the generation and conduction of nerve impulses signal pain, it is also used in obstetrics and some times for relief pain in the lower back and tooth extraction [2].Several methods have been reported for the determination of procaine HCl include High performance liquid chromatography [3,4], differential-plus voltammetry and electrochemichal analysis [5-8], electrophoresis [9,10], atomic absorption [11], ion association titration [12], gas chromatography [13], fluorimetry [14-16], sequential injection analysis [17,18], flow injection analysis [19,20], chemiluminsense [21], colorimetry and spectrophotometric methods [22][23][24][25][26][27][28].The present study describes the development of batch and FIA methods based on diazotiazation and coupling reaction between diazotized Procaine HCl and chromatropic acid reagent in alkaline medium.The pink product has been spectrophotometrically measured at 508 nm.The analytical procedure is simple, fast, accurate, and has been applied for the determination of Procaine HCl in pure and injections preparations using standard addition method.The reaction can be carried out in batch and FIA and the two approaches are compared.

Materials and Methods:
Apparatus All spectral and absorbance measurements were carried out on a Shimadzu UV-Visble-260 digital double-beam recording spectrophotometer (Tokyo-Japan), and photomech 301-D + spectrophotometer (Optima-Japan) using 1-cm quartz cells.A quartz flow cell with 50 µl internal volume and 1 cm bath length was used for the absorbance measurements.
A two channel manifold (Figure 4) was employed for the FIA spectrophotometric determination of Procaine HCl.A peristaltic pump (Ismatec, Labortechnik-Analytic, CH-8152, Glatbrugg-Zurich, Switzerland, six channels) was used to transport the reagents solutions.Injection valve (Rheodyne, Altex 210, Supelco-USA) was employed to provide appropriate injection volumes of standard solutions and samples.Flexible vinyl tubing of 0.5 mm internal diameter was used for the peristaltic pump.Reaction coil (RC) was of Teflon with internal diameter of 0.5 mm.In (Figure 4) the chromatropic acid (R1) stream was combined with injected sample (Procaine HCl and sodium nitrite and hydrochloric acid ) and they merged with NaOH (R2) stream at T-link then mixed in reaction coil (RC) with length of 100 cm, injection loop of (150 μl), total flow rate of 2.4 ml/min, the absorbance was measured at 508 nm. and at temperature (10-15° C).

Reagents and materials
Analytical reagents grade chemicals and distilled water were used thoroughly.

a-For batch method
Procaine HCl stock solution (1000 µg.ml -1 = 3.66 x 10 -3 M): a 0.10000 gm amount of pure Procaine HCl (SDI) was dissolved in amount of distilled water then complete to 100 ml in a volumetric flask with the same solvent.Procaine HCl working solution (500 µg.ml -1 = 1.832 x 10 -3 M), was prepared by dilution of 50 ml of the stock solution to 100 ml volumetric flask with distilled water.Sodium nitrite (3.66 x 10 -3 M): A 0.02525 gm amount of NaNO 2 (Merck) was dissolved in a 100 ml volumetric flask with distilled water.More dilute solution (1.832 x 10 -3 M) was prepared by simple dilution.
Chromatropic acid (0.1 % w/v) reagent solution: prepared by dissolving 0.1 gm of pure chromatropic acid (BDH) in amount of water and complete the volume to 100 ml volumetric flask with distilled water.

Hydrochloric acid (BDH) (1M), Sodium hydroxide (2M).
b-For flow-injection method Diazotized Procaine HCl stock solution (500 µg.ml -1 =1.832 x 10 -3 M): a 0.0500 gm amount of pure Procaine HCl (SDI) was dissolved in an amount of distilled water then 0.01264 gm of sodium nitrite and 8 ml of (1M) Hydrochloric acid were added, shake well and complete to 100 ml in a volumetric flask with distilled water.
Sodium hydroxide (0.7M)More dilute solutions were prepared fresh daily by suitable dilution of the stock solution with distilled water.

Procedure for Injections:
Two types of injection were analyzed by the developed methods, these include:-1-Procaine benzyl penicillin injection (300 mg Procaine penicillin)-Ajanta House Charkop-India 2-Procaine benzyl penicillin injection-(800 mg Procaine Penicillin)-Troge Medical GMBH -Germany For these types of injection, an accurately weighed portion from mixed three vials powder, equivalent to about 0.0500 gm of Procaine HCl, was transferred into a 100 ml volumetric flask and was dissolved and completed to the mark with distilled water to obtained 500 µg.ml -1 .Aliquot samples were treated exactly the same as in the procedure cited for the batch method.
For FIA method, an accurately weighed portion from mixed three vials powder equivalent to about 0.0500 gm of Procaine HCl, 0.01264 gm of sodium nitrite and 8 ml of (1M) Hydrochloric acid were added, shake well and complete to 100 ml in a volumetric flask with distilled water to obtained 500 µg.ml -1 (1.832 x 10 -3 M).More dilute solutions were prepared by simple dilution for the stock solution with distilled water.

Procedures: General batch procedure
An increasing volumes (0.05-2 ml) of 500 µg.ml -1 (1.832 x 10 -3 M) Procaine HCl was transfer into a series of 25 ml standard flask.(0.05-2 ml) of sodium nitrate (1.832 x 10 -3 M) and 2 ml of 1M Hydrochloric acid were added and shake well followed by the addition of 3 ml of (0.1%) chromatropic acid and 1 ml of sodium hydroxide (2 M).The contents of the flasks were diluted to the mark with distilled water, mixed well and left for 10 min at temperature (10-15 o C), the absorbance of the pink dye formed was measured at 508 nm against a reagent blank containing all materials except Procaine HCl.A calibration graph was drawn and the regression equation was calculated, for the optimization of conditions and in all subsequent experiments, a 1 ml of (500 µg in a final volume of 25 ml = 1.832x10 -3 M) of Procaine HCl was used.

General FIA procedure
A diazotized Procaine HCl solution in the range of 5-400 µg.ml -1 were prepared from the working solution of 500 µg.ml -1 (1.832x10 -3 M) and stoichiometric amount of sodium nitrite.A 150 µl portion of Procaine HCl was injected into the stream of the chromatropic acid (0.07% w/v) then the mixture combine with (0.7 M) NaOH at T-link with a total flow rate of 2.4 ml min -1 for the two channels, the resulting absorbance of the red product was measured at 508 nm and a calibration graph was constructed.Optimization of conditions were carried out on 500 µg.ml -1 of Procaine HCl.

Batch spectrophotometric determination
The factors that affecting on the sensitivity and stability of the colored product resulting from the diazonium reaction between diazotized Procaine HCl and chromatropic acid in alkaline medium were carefully studied.A typical spectrum for the azo dye formed was measured versus reagent blank which has negligible absorbance at λmax 508 nm (Figure 1).

Fig. 1: Absorption spectra of the azo dye (20 ppm) against reagent blank (A) and blank against distilled water (B).
The experimental conditions for the determination of Procaine HCl were established.The diazonium reaction occurred in an acidic medium and hydrochloric acid of concentration 1M was selected, the effect of different volumes of 1 M of HCl were studied and 2 ml volume seems to be the optimum for an intense azo dye absorbance.A 1:1 mole ratio of Procaine HCl to sodium nitrite of (1.83x10 -3 M) was used in order to prevent the effect of excess of sodium nitrite.The effect of coupling reagent (0.1% w/v) Chromatropic acid volume (0.3-5 ml) on the intensity of the absorbance, were studied and 3 ml was found to be optimum.The absorbance of the dye formed became more intense and stable in alkaline medium, therefore, the effect of different alkaline solutions on the colored product were studied sodium hydroxide, ammonium hydroxide, potassium hydroxide, sodium acetate and sodium carbonate.Maximum sensitivity and stability were obtained only when the reaction was carried out in the presence of sodium hydroxide solution.The effect of different concentrations of NaOH (0.1-3 M) were studied, and 2 M seems to be optimum.The effect of (2M) NaOH volumes (0.1 to 2.5 ml) were also studied and 1 ml was found optimum.Experimental results revealed that the colour intensity reach a maximum after Procaine HCl solution had been reacted with chromatropic acid in alkaline medium for 20 min, therefore, a 20 min development time was suggested as the optimum reaction time and remain stable for 120 min.The order of addition of the reagents is an essential part of the experiment, it was found that the order of addition of the reagent cited under general procedure gave a maximum color intensity and the minimum absorbance of the blank and was used in all subsequent experiments.The effect of temperature on the colour intensity of the dye was studied.In practice, a higher absorbance was obtained when the reaction was developed at room temperature (10-15 o C) and when the calibrated flasks were placed in an ice bath (0-5 o C) or in water bath (60 o C) a decreased in absorbance were observed.The stoicheiometry of the reaction between Procaine HCl and chromatropic acid was investigated by using both continuous variation and molar ratio methods respectively.The result obtained (figure 2&3) show that a (1:1) azo dye was formed between diazotized procaine HCl and chromotropic acid

Fig. 3: Continuous variation plot of the reaction between diazotized Procaine HCl and chromatropic acid (1.38x10 -3 M)
A reaction subsequent based on the above results is shown in Scheme (1).

Scheme (1): reaction sequence
The product formed was soluble in water.The apparent stability constant was calculated by comparing the absorbance of a solution containing stiochiometric amount of diazotized Procaine HCl (1.832x10 -3 M) and chromatropic acid (1.83x10 -3 M) (A S ) with that of a solution containing a five fold excess of chromatropic acid reagent (A m ) and according to analytical procedure.The average stability constant (K)= 7.528x10 4 L.mol -1 , where is [K=(1-α)/ α 2 C] and α=A m -A s /A m [29].The regression equation obtained, and the analytical features of the procedure are summarized in (Table 1).It is also summarized the main performance of the flow procedure developed for Procaine HCl determination in order to make an effective comparison between the two approach.

FIA-spectrophotometric determination
The batch method for the determination of Procaine HCl was adopted as a basis to develop FIA procedure.The manifold used for the determination of Procaine HCl was designed to provide different reaction conditions for magnifying the absorbance signal generated by the reaction of the diazotized Procaine HCl with chromatropic acid in sodium hydroxide medium.Maximum absorbance intensity was obtained when the sample (diazotized Procaine HCl 500 µg.ml -1 ) was injected into a stream of chromatropic acid and then mixed with sodium hydroxide as given in (Figure 4).The influence of different chemical and physical FIA parameters on the absorbance intensity of the colored product was optimized as follows:

Optimization of chemical parameters
The effect of various concentrations of Hydrochloric acid (0.1-4M) were studied for the formation of diazotized Procaine HCl in the presence of sodium nitrite, 1M HCl seems to be optimum as shown in (Figure 5).

Fig. 5: The effect of the concentration of hydrochloric acid in (M)
The effect of various concentrations of chromatropic acid was investigated.A concentration of (0.07% w/v) chromatropic acid, gave the highest absorbance and was chosen for further experiments as shown in (Figure 6).It was observed that the reaction between diazotized Procaine HCl and chromatropic acid depends on alkaline medium, therefore the effect of different concentrations of sodium hydroxide was studied and 0.7M was found to be the optimum as shown in (Figure 7).The effect of total flow rate on the sensitivity of the colored reaction product was investigated in the range of 0.6-4ml min -1 .The results obtained showed that a total flow rate of 2.4 ml min -1 , (1.2 ml min -1 in each line) gave the highest absorbance as shown in (Figure 8), and was used in all subsequent experiments.Flow rate (ml/min) Abs.

Fig. 8: The effect of the total flow rate (ml/min)
The volume of the injection sample was varied between 50-250 µl using different length of sample loop.The results (Figure 9) obtained showed that injected sample of 150 µl gave the best absorbance.

Sample loop
Abs.

Fig. 9: effect of the injection loop (µl)
The coil length is an essential parameter that affects on the sensitivity of the colored reaction product and was investigated in the range of 25-250 cm.The results obtained showed that a coil length of 100 cm gave the highest absorbance as shown in (Figure 10) and was used in all subsequent experiments.

Fig. 10: The effect of the length of the reaction coil in (cm)
The reaction time is also an important parameter that affected on the sample throughput and was investigated by calculating the interval time between the sample injection and the appearance of the end of the signal.The reaction time of each sample was 51 sec, therefore the sample through put was 70 samples per hour. (µl)

Analytical characteristics
Analytical characteristics such as linear range, detection range, correlation coefficient and relative standard deviation (RSD) of each method were determined under the optimized conditions that are shown in (Table 1).In comparison of the batch with the FIA procedure, the later is more convenient than the former method because of its speed (sample through-put of 70 injection h -1 ), wider linear range of calibration graph, and good recovery were obtained.

Analysis of pharmaceutical products (injection samples)
The standard addition method was applied for the quantitative determination of Procaine HCl in injections.Two types (different origins) of injections preparations containing Procaine HCl in procaine penicillin were analyzed and as shown in (Table 2).It was found that, when the proposed method was applied directly to the determination of procaine HCl in injections , a high recoveries were obtained, this might be due to the interference from benzyl penicillin that present in injections.Therefore, a standard additions method was applied (Figure 11 & 12) which involves adding increment volumes (0-7 ml) of a standard solution of 100 µg.ml -1 and (0-5 ml) of standard solution of 60 µg.ml -1 of diazotized Procaine HCl to a fixed volume sample (0.2 ml of 500 µg.ml -1 and (1.5 ml of 40 µg.ml -1 ) of diazotized pharmaceutical preparations for both batch and FIA methods and employing the conditions described under procedure .They gave a good accuracy and precision (Table 2).The proposed method was compared successfully with the British pharmacopeia's standard method [1].

Conclusion:
Batch and FIA spectrophotometric method were proposed for the determination of Procaine HCl in pure and in pharmaceutical preparations.These methods have the advantage of simplicity, speed, accuracy and the use of inexpensive equipment.The speed of the proposed methods and its precision make them suitable for the quality control of formulations containing this drug.The wide linear range of the proposed methods that obeyed Beer' law gave a good application for the pharmaceutical preparation, and can be used as a reliable and advantageous alternative to the other previously explorted methods for routine analysis of Procaine HCl in pharmiceutical samples.
Fig. 6: The effect of the concentration of chromatropic acid reagent in (0.7M) of NaOH Fig. 7: The effect of the concentration of sodium hydroxide in (M)

Fig
Fig. 11: the standard addition method For the batch method

Table 2 : Application of the standard addition method for both batch and FIA and official methods for the determination of Procaine HCl.
*For five determinations